Ultrasensitive Immunoassay Based on Anodic Near-Infrared

Nov 19, 2012 - (BSA) were obtained from Shanghai Linc-Bio Science Co. Ltd. (China). All chemicals used in the experiments were of analytical grade or ...
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Ultrasensitive Immunoassay Based on Anodic Near-Infrared Electrochemiluminescence from Dual-Stabilizer-Capped CdTe Nanocrystals Guodong Liang, Shufeng Liu, Guizheng Zou,* and Xiaoli Zhang School of Chemistry and Chemical Engineering, Shandong University, Jinan, 250100, China S Supporting Information *

ABSTRACT: A sandwich-typed near-infrared (NIR) electrochemiluminescence (ECL) immunoassay was developed with dual-stabilizer-capped CdTe nanocrystals (NCs) as ECL labels and α fetoprotein antigen (AFP) as model protein. The dualstabilizer-capped NIR CdTe NCs were promising ECL labels because of their NIR ECL emission of 800 nm, low anodic ECL potential of +0.85 V, and high biocompatibity, which can facilitate interference-free and highly sensitive ECL bioassays. Upon the immunorecognition of the immobilized AFP to its antibody labeled with dual-stabilizer-capped CdTe NCs, the proposed immunoassay displayed increasing ECL intensity, leading to a wide calibration range of 10.0 pg/mL to 80.0 ng/mL with a detection limit of 5.0 pg/mL [signal-to-noise ratio (S/N) = 3] without coupling any signal amplification procedures. The NIR ECL immunoassay for real samples displayed very similar results with those of Ru(bpy)32+ reagent kit based commercial ECL immunoassay, which not only proved for the efficiency of NIR ECL from dual-stabilizer-capped CdTe NCs but also paved the road for development of novel ECL emitters and corresponding reagent kits. ear-infrared (NIR) fluorescence has attracted much more interests in biomedical and diagnostic fields because NIR emission offers several advantages, including improved tissue penetration, lower background interference, and reduced photochemical damage. 1−7 Semiconductor nanocrystals (NCs) were promising candidates for labeling in NIR fluorescent sensing because of their size-dependent photoluminescence (PL) and stable photochemistry.3,8 However, photobleaching and autofluorescence are still unavoidable limits in NIR fluorescence-based bioanalysis, due to the photoexcitation nature of fluorescent techniques. Electrochemiluminescence (ECL) is superior to fluorescence in terms of sensitivity and signal-to-noise ratio due to absence of background from unselective photoexcitation.9−11 Typically, the commercial ECL detection system based on Ru(bpy)32+/ tri-n-propylamine (TPrA) reagent kits has been widely used in biomedical and diagnostics assays. However, the ECL emissions from traditional emitters, such as luminol and Ru(bpy)32+, are mainly located in visible ranges. The NIR ECL analysis could probably provide an alternative to both the NIR fluorescent bioassay and the traditional ECL bioassays. Although some NCs were promising ECL emitters,12−22 the NCs-based ECL was mainly limited in the visible region.23−31 Recently, a strategy based on NIR ECL using NCs has been designed for the detection of target antigen with dual-signal amplification.19 However, NIR ECL is still far from practical applications until now. It is believed that there are three kinds of limitations for the bioassay of NCs-based NIR ECL. First, NIR ECL emissions

N

© 2012 American Chemical Society

were usually involved in low ECL intensity22 or high ECL triggering potentials.19−21 Second, NIR ECL emissions were mainly generated at cathode using persulfate as coreactant.19−21 The hydrogen peroxide, generated with the electrochemical reduction of dissolved oxygen, was also a cathodic ECL coreactant and may cause unexpected interference.31 Finally, current NIR ECL detection was carried out at the wavelength of around 700 nm, which could not effectively meet the requirement of interference-free sensing.19,20 Therefore, novel NIR ECL emitters are highly desired for practical applications of ECL detection. Recently, our group has developed a convenient one-pot synthetic strategy for preparation of dual-stabilizer-capped NIR CdTe NCs with strong anodic ECL emissions.32−34 Herein, the dual-stabilizer-capped CdTe NCs were used as NIR ECL labels for a sandwich-typed NIR ECL immunoassay with 2(dibutylamino)ethanol (DBAE) as coreactant16,35 and α fetoprotein (AFP) antigen as model protein, as shown in Scheme 1. Because of the NIR ECL emission of 800 nm, low anodic ECL potential of +0.85 V, and high biocompatibity of ECL labels, the proposed NIR immunoassay demonstrated acceptable performance for determining AFP in real samples. Received: August 4, 2012 Accepted: November 19, 2012 Published: November 19, 2012 10645

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added to the above solution and the crude materials turned to green-yellow immediately. After the resulting mixture was refluxed at 100 °C for 10 min, 2.4 mL of N2H4·H2O was injected into the above solution, and then the final mixed solution was refluxed under open-air conditions for 25 h to obtain water-soluble CdTe NCs. The obtained crude products were precipitated for three times by acetone with centrifugation at 12 000 rpm for 10 min, and the resultant precipitates were redissolved in double-distilled water as stock solution and then kept under dark at 4 °C. The concentration of the NCs stock solution was estimated at 0.32 μmol/L with an empirical equation.36 Preparation of NCs−Ab2 Conjugates. An amount of 120 μL of 0.32 μmol/L CdTe NCs was transferred into 240 μL of 0.10 mol/L pH 6.0 PBS containing 100 mg/mL EDC and 100 mg/mL NHS for 30 min at 37 °C. The mixed solution was then centrifuged and the resultant precipitates were redispersed in 10.0 mmol/L pH 7.4 PBS containing 25.0 μg/mL Ab2 for 12 h at 37 °C. An amount of 40 μL of 3% BSA solution was then introduced in the mixed solution to block the nonspecific binding sites of the NCs/Ab2 conjugates.37 The final solution was then purified by centrifugation and the NCs/Ab2 conjugates were dispersed with 80 μL of 10.0 mmol/L pH 7.4 PBS and stored at 4 °C for further use. Preparation of the ECL Immunosensor. The fabrication procedure of the NIR ECL immunosensor is shown in Scheme 1. The 5.0 mm Au disk electrodes were prepolished with 0.3 μm alumina slurry and washed with water, then cleaned with freshly prepared piranha solution (98% H2SO4/30% H2O2, 7:3, v/v) for ∼10 min, followed by washing with copious amounts of distilled water and ethanol. After being dried in a stream of N2, the Au electrodes were transferred into 20.0 mmol/L TGA ethanol solutions for 12 h. The newly formed Au/TGA was rinsed with ethanol and water and dried with a stream of nitrogen. The carboxyl group of Au/TGA was then activated in 0.10 mol/L pH 6.0 PBS containing 100 mg/mL EDC and 100 mg/mL NHS for 30 min. After being washed thoroughly with 10.0 mmol/L pH 7.4 PBS, the Au/TGA electrodes were transferred into 10.0 mmol/L pH 7.4 PBS containing 20.0 μg/ mL anti-AFP (Ab1) for 3 h at 37 °C. The electrodes were then washed with PBS and blocked with 3% BSA at 37 °C for 1 h. Finally, the electrodes were incubated in AFP solution for 3 h at 37 °C and NCs/Ab2 (or Ab2) solution for 3 h at 37 °C, respectively, to obtain sandwich-type immune complex at the Au disk electrode surface (Au/TGA/Ab1−Ag−Ab2−NCs). AFP Detection in Serum Samples. For the preparation of serum, 1.0 mL of human blood (provided by Qilu Hospital, Shandong University) from an outpatient was collected in a sample tube. The serum was separated after putting the sample in an incubator at 37 °C for 30 min to remove the red cell. The above serum layer was centrifuged at 1100 rpm for 6 min. The resultant human serum sample was then stored at −20 °C until use. For the detection in serum matrix, newly obtained independent serum samples were then used to prepare the immunosensor. The obtained independent serum samples with higher AFP levels were diluted properly with 10.0 mmol/L pH 7.4 PBS before NIR ECL measurement. To evaluate the performance of the NIR ECL immunosensor, the AFP concentrations of outpatients were also determined by Ru(bpy)32+/TPrA-based commercial reagent kit on Roche Cobas E601 by the bioanalytical laboratory of Qilu Hospital.

Scheme 1. Schematic Representation of Preparations of NIR Sandwich-Typed Immunoassay

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EXPERIMENTAL SECTION Materials. Cadmium chloride (CdCl2·2.5H2O) was purchased from Alfa Aesar China Ltd. (China). Mercaptopropionic acid (MPA) was obtained from Aldrich Chemicals (U.S.A.). Na2TeO3 was obtained from Shanghai Jingchun reagent Co., Ltd. (China). Sodium hexametaphosphate (HMP) was obtained from Guangcheng Chemical Co., Ltd. (China). Hydrazine hydrate was obtained from Sinopharm Chemical Reagent Co., Ltd. (China). DBAE was purchased from Sigma Chemical Co. (MO, U.S.A.). Thioglycolic acid (TGA) was obtained from Sinopharm Chemical Reagent Co., Ltd. (China). N-Hydroxysuccinimide (NHS) was obtained from Huifeng Chemical Industry Ltd. (China). 1-Ethyl-3-(3-dimethyl-aminopropyl) (EDC) carbodiimide hydrochloride was obtained from Shanghai Medpep Co. Ltd. (China). AFP (Ag), monoclonal antibody to AFP (Ab1 and Ab2), and bovine serum albumin (BSA) were obtained from Shanghai Linc-Bio Science Co. Ltd. (China). All chemicals used in the experiments were of analytical grade or the highest purity grade (commercially available) and used without further purification. All solutions were prepared with doubly distilled water. Apparatus. The absorption spectrum was obtained with a TU-1901 UV−vis spectrophotometer (Beijing, China). PL was recorded with a WGY-10 spectrofluorimeter (Tianjin, China). X-ray photoelectron spectra (XPS) were obtained with a Kα Xray photoelectron spectrometer (Thermo Fisher Scientific Co., U.S.A.). Cyclic voltammetry and ECL measurements were carried out with an MPI-A multifunctional electrochemical and chemiluminescent system (Xi’an Remex Analytical Instrument Ltd. Co., China). All ECL measurements were performed in a self-made glass cell composed of a Au disk electrode (5.0 mm in diameter), a platinum counter electrode, and a Ag/AgCl (saturated KCl) reference electrode. The ECL spectrum was recorded with an optical multichannel analyzer (SpectraPro300i, Acton Research Co., Acton, MA, U.S.A.) coupled with a CHI 822 analyzer (CH Instrument, Shanghai, China). Preparation of CdTe NCs. The dual-stabilizer-capped CdTe NCs were prepared in aqueous medium according to previously reported one-pot synthetic strategy.32 An amount of 1.60 mL of cadmium chloride solution (CdCl2, 0.10 mol/L) was added to a three-necked flask containing 50 mL of H2O, and then HMP (293.6 mg) and MPA (34.6 μL) were added successively under magnetic stirring. After the solution pH was adjusted to 8.0 by 1.0 mol/L NaOH, Na2TeO3 (5.3 mg) was 10646

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+1.2 V)10,11 and the other CdTe NCs (about +1.17 V) based immunoassay,29 so the proposed ECL immunoassay can efficiently avoid the electrochemical interference. The ECL spectrum of Au/TGA/Ab1−Ag−Ab2−NCs also demonstrated further a red-shifted emission peak at around 800 nm (inset in Figure 1, curve c1) to that of the reported NIR ECL immunosensor at around 700 nm;19 thus, the proposed ECL immunoassay can efficiently avoid the interference caused by biological tissues. So, improved ECL immunoassay performance was expected with the dual-stabilizer-capped CdTe NCs as labels. We suppose the ECL mechanism of the dual-stabilizercapped CdTe NCs/DBAE system as the following equations:16

RESULTS AND DISCUSSION NIR ECL Behaviors of the NIR Immunosensor. The dual-stabilizer-capped CdTe NCs with PL at 780 nm (inset in Figure 1, curve b1) were chosen as ECL labels due to their NIR

(CdTe)NCs → (CdTe)NCs•+ + e−

(1)

DBAE → DBAE•+ + e−

(2)

DBAE•+ → DBAE• + H+

(3)

(CdTe)NCs•+ + DBAE• → (CdTe)NCs * + DBAE fragments (CdTe)NCs * → (CdTe)NCs + hν Figure 1. (A) ECL and (B) electrochemical behavior of (a) bare Au, (b) Au/TGA, (c) Au/TGA/Ab1−Ag−Ab2, and (d) Au/TGA/Ab1− Ag−Ab2−NCs in 0.10 mol/L pH 9.2 PBS containing 20.0 mmol/L DBAE at a scan rate of 100 mV/s. Inset: (a1) absorbance, (b1) PL of NIR-emitting CdTe NCs, and (c1) ECL spectrum of Au/TGA/Ab1− Ag−Ab2−NCs in 0.10 mol/L pH 9.2 PBS containing 20.0 mmol/L DBAE at a scan rate of 100 mV/s. Ag concentration: 40.0 ng/mL.

(4) (5)

Effects of Label Ratios on the Performance of the NIR Immunosensor. It is well-known that high label ratio can lead to high sensitivity in ECL immunoassay.40−42 As shown in Figure 2, with increasing label ratios, the ECL intensity of the

emission feature. Because the competitive binding behaviors of MPA and HMP toward Cd2+ ions at the surface of the CdTe NCs resulted in improved PL features,32 the PL quantum yield of the NCs was determined as around ∼10%. The sharp excitonic peak in absorption and the narrow PL spectrum (inset in Figure 1, curves a1 and b1) also indicated their superior absorption and light emission features.32−34 The molar ratio of Cd/S/P/Te atoms at the NC surface was calculated to be 43.51/35.71/14.76/6.00 by the XPS method (XPS pattern is given as Figure S1 in the Supporting Information). According to previously published works,32 the chemical structure of NCs was deduced as a CdTe core which was stabilized with MPA and HMP outside. DBAE was chosen as coreactant because it was environmentally friendly and could be involved in lowpotential ECL emission from NCs.16 As shown in Figure 1, no ECL emission was recorded from bare Au (curve a), Au/TGA (curve b), and Au/TGA/Ab1−Ag−Ab2 (curve c) in 0.10 mol/ L pH 9.2 PBS containing 20.0 mmol/L DBAE, whereas Au/ TGA/Ab1−Ag−Ab2−NCs, the sandwich immune complex formed on the Au electrode with ECL labels, displayed strong anodic ECL at about 0.85 V (curve d), because dual-stabilizercapped CdTe NCs could undergo a low-potential holeinjection process for ECL emission.34 The ECL behaviors of the ECL labels in the sandwich immune complex were similar to those of the bare CdTe NCs synthesized by the same method,34 which indicated the immunorecognition of the immobilized AFP to the CdTe NCs labeled Ab2 can be utilized for ECL immunoassay. It has been reported that, in order to avoid undesirable anodic reactions, lower oxidation potential is normally desired when Ru(bpy)32+ or its derivatives are used as ECL labels in biosystems.38,39 In our approach, the ECL peak potential of Au/TGA/Ab1−Ag−Ab2−NCs was much lower than the ECL peak potential from Ru(bpy)32+ systems (beyond

Figure 2. ECL behavior of Au/TGA/Ab1−Ag−Ab2−NCs with NCs/ Ab2 label ratio of (a) 0.5:1, (b) 1:1, (c) 2:1, (d) 3:1, and (e) 4:1 in 0.10 mol/L pH 9.2 PBS containing 20.0 mmol/L DBAE at a scan rate of 100 mV/s. Inset: effects of label ratio on the ECL intensity. Ag concentration: 40.0 ng/mL.

NIR immunosensor increased gradually and reached maximum intensity at a NCs/Ab2 ratio of 3:1 (inset in Figure 2). Such results not only proved the excellent biocompatible feature of NIR CdTe NCs but also suggested an optimized label ratio can be achieved with the dual-stabilizer-capped CdTe NCs. The decreased ECL response at higher NCs/Ab2 ratios was probably due to the fact that NCs could block the active sites of Ab2 in immunoreaction processes. The NCs/Ab2 ratio of 3:1 was chosen for following experiments. Effects of Scan Rate on the Performance of the NIR Immunosensor. Figure 3 shows the effects of scan rate on the ECL profiles of the proposed NIR immunosensor. With the increase of the scan rate from 10 to 200 mV/s, the ECL peak potential gradually increased from around 0.81 to 0.86 V. With 10647

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of 3, which was much lower than that with Ru(bpy)32+43,44 or the other NCs-based45,46 ECL sensing method. Reproducibility of the NIR immunosensor for AFP determination was estimated with seven immunosensors made at Au electrodes, and the ECL intensity of the immunosensors demonstrated a relative standard derivation of 3.6% with AFP at 5.0 ng/mL level (as shown in Figure 5).

Figure 3. ECL behavior of Au/TGA/Ab1−Ag−Ab2−NCs in 0.10 mol/L pH 9.2 PBS containing 20.0 mmol/L DBAE at a scan rate of (a) 10, (b) 50, (c) 100, (d) 150, and (e) 200 mV/s. Inset: effects of scan rate on the ECL intensity. Ag concentration: 40.0 ng/mL.

the increase of scan rate from 10 to 100 mV/s, the ECL intensity of Au/TGA/Ab1−Ag−Ab2−NCs also increased and reached the maximum emission intensity at 100 mV/s. The ECL intensity decreased with the further increase of the scan rate from 100 to 200 mV/s, which was probably due to the high scan rate being unfavorable for the hole injection of the ECL label and the oxidation of coreactant at the electrode surface. Previous research concerning the ECL feature of bare dualstabilizer-capped CdTe NCs also displayed similar results.34 The scan rate of 100 mV/s was chosen in the following NIR ECL assays. NIR ECL Detection of AFP. Under optimal conditions, the sandwich-typed immunoassay was applied for the detection of AFP. Figure 4 depicts the ECL profiles for the NIR

Figure 5. Reproducibility of the NIR ECL immunosensor in 0.10 mol/ L pH 9.2 PBS containing 20.0 mmol/L DBAE with AFP at 5.0 ng/mL level at a scan rate of 100 mV/s.

AFP Sensing in Human Serum. To further investigate the feasibility of the immunosensor for practical applications, the sensing of AFP in real human serum was carried out. The samples were determined with Ru(bpy)32+/TPrA diagnostic reagent kit based commercial ECL test on Roche Cobas E601 at the same time. Table 1 lists the results of three independent Table 1. AFP Content in Three Human Serum Samples Determined by the Proposed Immunosensor and Commercial Reagent Test Kit sample a

CdTe/DBAE (ng/mL) Ru(bpy)32+/TPrA (ng/mL)b

1

2

3

140.0 166.0

42.0 43.8

5.3 5.7

a The proposed immunosensor. bRu(bpy)32+/TPrA-based commercial reagent test.

serum samples obtained using the proposed immunoassay and corresponding commercial ECL test. The concentration of AFP obtained with the proposed immunoassay was close to that obtained with the commercial ECL test on Roche Cobas E601. All these results not only indicated the acceptable accuracy of the proposed NIR ECL immunoassay for the detection of AFP in clinical samples but also demonstrated that the dualstabilizer-capped CdTe NCs can be utilized for the development of novel ECL reagent kits in the NIR region.

Figure 4. ECL behavior of Au/TGA/Ab1−Ag−Ab2−NCs in 0.10 mol/L pH 9.2 PBS containing 20.0 mmol/L DBAE with (a) 0.0, (b) 0.0050, (c) 0.010, (d) 0.10, (e) 1.0, (f) 40.0, and (g) 80.0 ng/mL AFP at a scan rate of 100 mV/s. Inset: effects of AFP concentration on the ECL intensity.

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CONCLUSIONS In conclusion, an ultrasensitive NIR ECL immunoassay was developed by using dual-stabilizer-capped CdTe NCs as ECL labels with the advantages of their NIR ECL emission window at 800 nm, low oxidation potential, and high biocompatibility. The immunoassay can be used to detect AFP antigen in serum samples with similar capacity of the Ru(bpy)32+ reagent kit based commercial ECL test. This proposed immunoassay not only provided a promising alternative for NCs-based NIR PL bioassay strategy but also opened a new way for designing a

immunosensor after reacting with different concentrations of AFP. As can be seen, the ECL signal increased with the increase of concentrations of AFP as the consequence of the efficient capture of the AFP by sandwich immunoassay. A linear relation between ECL intensity and the logarithm of AFP concentration was obtained from 10.0 pg/mL to 80.0 ng/mL (R = 0.994) with the detection limit of 5.0 pg/mL at a signal-to-noise ratio 10648

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novel NIR ECL reagent kit with practical applications. Moreover, the difference between the ECL spectrum of the dual-stabilizer-capped CdTe NCs and that of Ru(bpy)32+ might enable the development of ECL-based multiplexing assay.

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ASSOCIATED CONTENT

S Supporting Information *

XPS pattern of the dual-stabilizer-capped CdTe NCs. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Author Contributions

The first two authors contributed equally to this work. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This project was supported by the National Natural Science Foundation of China (Grant Nos. 20705016 and 20975061) and the Independent Innovation Foundation of Shandong University (Grant No. 2012TS002). We are also grateful for the outpatient’s agreements of serum sample and the support of the bioanalytical laboratory from Qilu Hospital of Shandong University.

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dx.doi.org/10.1021/ac302236a | Anal. Chem. 2012, 84, 10645−10649